Cinnamaldehyde alleviates doxorubicin-induced cardiotoxicity by decreasing oxidative stress and ferroptosis in cardiomyocytes

Although doxorubicin (DOX) is an efficient chemotherapeutic drug for human tumors, severe cardiotoxicity restricts its clinical use. Cinnamaldehyde (CA), a bioactive component isolated from Cinnamonum cassia, possesses potent anti-oxidative and anti-apoptotic potentials. The major aim of this study was to evaluate the protective role of CA against DOX-induced cardiotoxicity. To this end, cardiomyocyte injury models were developed using DOX-treated H9c2 cells and DOX-treated rats, respectively. Herein, we found that CA treatment increased cardiomyocyte viability and attenuated DOX-induced cardiomyocyte death in vitro. CA further protected rats against DOX-induced cardiotoxicity, as indicated by elevated creatine kinase (CK) and lactate dehydrogenase (LDH) levels, myocardium injury, and myocardial fibrosis. CA alleviated DOX-induced myocardial oxidative stress by regulating reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels. Mechanistically, CA markedly accelerated nuclear translocation of nuclear erythroid factor 2-related factor 2 (Nrf2) and increased heme oxygenase-1 (HO-1) expression. Consequently, CA decreased DOX-induced cardiomyocyte ferroptosis, while Erastin (a ferroptosis agonist) treatment destroyed the effect of CA on increasing cardiomyocyte viability. Taken together, the current results demonstrate that CA alleviates DOX-induced cardiotoxicity, providing a promising opportunity to increase the clinical application of DOX.

CA, a bioactive component isolated from Cinnamonum cassia, possesses potent anti-oxidative [27,28], anti-inflammatory [29], and anti-tumor capabilities [30].Huang et al. demonstrated that CA increases phase II detoxifying enzyme expression by activating Nrf2 in hepatocellular carcinoma cells [27].Kim et al. reported that CA alleviates dental pulp cell oxidative stress via activating the Nrf2/HO-1 pathway [28].In the study, the biological effect of CA on alleviating DOX-induced cardiotoxicity was evaluated in vitro and in vivo.

Cell culture and treatment
H9c2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultivated in DMEM (GIBCO, NY, USA) containing 10% FBS (GIBCO) in a humidified CO2 incubator at 37 ˚C.H9c2 cells were seeded into 96-well plates (5000 cells per well) or 6-well plates (2 × 10 6 cells per well) overnight and then treated with DOX (0.5, 1, 2, 4, 8, and 10 μM) for 24 h.

Rat model of DOX-induced myocardial injury
The protocol was performed with the approval of the Experimental Animal Committee of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (No. PZSHUTCM210312009) according to ARRIVE guidelines [31] to decrease animals suffering.Male Sprague-Dawley rats (approximately 8 weeks old) were obtained from the Shanghai Model Organisms Center, Inc. (China) and maintained in specific pathogen-free facilities (temperature: 21-24˚C) with water and food ad libitum.In the DOX group, rats (n = 5) were injected intraperitoneally (i.p.) with DOX at doses of 15 mg/kg (DOX was dissolved in 0.9% saline) as previously described [32].In the CA treatment group, after treatment with 15 mg/kg of DOX, rats (n = 5) were additionally gavaged with CA at a dose of 50 mg/kg for 6 weeks, as previously described [33,34] and in our preliminary experiments.In the control group (n = 5), rats were injected i.p. with 0.9% saline.Subsequently, rats were euthanized by inhalation of 2% isoflurane to collect blood and heart tissues for subsequent analysis.

TUNEL assay
TUNEL was used to assess H9c2 cell death.In brief, cells were seeded into 12-well plates and then treated with 4 μM of DOX and 100 μM of CA.After treatment for 24 h, cells were fixed with 4% PFA and stained with TUNEL (Beyotime, Shanghai, China) for 60 min.The fluorescence signal was captured with a XSPY-3201LED fluorescent microscope (CSOIF, Shanghai, China).

Serum biochemical indexes
Several serum biochemical indexes, including CK and LDH, were assessed using commercial kits (CK: ab155901, Abcam, CA, USA; LDH: ab102526, Abcam) in line with the manufacturer's specifications.

Intracellular ROS assay
H9c2 cells cultured in 12-well plates (0.1×10 5 cells / well) were treated with 4 μM of DOX and 100 μM of CA for 24 h.After washing thrice in PBS, cells were incubated with 10 μM of DCFH-DA (MedChem Express, NJ, USA) for 20 min in the dark.The fluorescence signal was captured with a XSPY-3201LED fluorescent microscope (CSOIF, Shanghai, China).

Assessment of MDA, SOD, GSH, and GSH-Px
Cardiac samples were homogenized in RIPA buffer on ice, and then the supernatant was collected from tissue lysates via centrifuging at 3000 rpm for 15 min at 4˚C.MDA, SOD, GSH, and GSH-Px levels in the supernatant were assessed using commercial kits in line with the manufacturer's specifications.

Quantitative real-time PCR (qRT-PCR)
Total RNA was collected with TRIzol (Beyotime), and first-strand cDNA was synthesized with M-MLV reverse transcriptase (Sigma-Aldrich, MO, USA) and Oligo (dT)18 primers.qRT-PCR was performed with BeyoFast™ SYBR Green qPCR Mix (Beyotime) on a StepOne-Plus real-time PCR system (Thermo Fisher Scientific).The temperature procedure is: 10 min at 95˚C followed by 35 cycles of 95˚C for 25 s and 58˚C for 10 s. β-actin was applied as a reference gene.The 2 (-ΔΔCT) method was applied to calculate the relative mRNA level [35].All primers were shown in S1 Table.

Statistics
The data were shown as the mean ± SD from three separate experiments.GraphPad Prism 7.0 (CA, USA) was used to compare the difference between two groups using the student's t-test or among multiple groups using a one-way ANOVA followed by the Scheffe ´test.The difference was statistically significant when p < 0.05.

CA alleviated DOX-induced myocardial injury
Consistent with previous studies [11,36], DOX exhibited a dose-dependent cytotoxic effect on H9c2 cells (Fig 1A).Based on the results, 4 μM of DOX was applied to treat H9c2 cells in subsequent experiments.The cytotoxic effects of CA on H9c2 cells were also assessed using the CA also alleviated DOX-induced H9c2 cell death (Fig 1D and 1E).Furthermore, DOX treatment increased serum CK and LDH levels in rats, whereas these effects were reversed by CA (Fig 1F and 1G).The results from HE staining showed that DOX resulted in an obvious cardiomyocyte injury, as suggested by misaligned muscle fibers and widened intercellular spaces, whereas these effects were blocked by CA (Fig 1H).Masson staining revealed that DOX increased collagen fibers in the myocardial interstitium, and their distribution was chaotic, whereas myocardial fibrosis was obviously improved and tended to normalize after CA treatment (Fig 1I).CA also decreased DOX-induced Col1α1 and α-SMA expression in heart tissues (Fig 1J).These results demonstrate that DOX exhibits serious cardiotoxicity in vitro and in vivo, which can be reversed by CA.

CA alleviated DOX-induced myocardial oxidative stress
Given that oxidative stress is a major mechanism of DOX-induced myocardial injury, we next investigated whether CA alleviated DOX-induced cardiotoxicity by decreasing oxidative stress.

CA activated Nrf2/HO-1 signaling
Nrf2 exerts a critical role in the anti-oxidative defense by increasing anti-oxidant enzyme expression [37,38], and CA possesses anti-oxidative capacity by promoting nuclear translocation of Nrf2 in human renal mesangial cells and dental pulp cells [28,39].Therefore, we further investigated whether CA alleviated myocardial oxidative stress by activating Nrf2/HO-1 signaling.Fig 3A-3C revealed that DOX markedly repressed nuclear translocation of Nrf2, as suggested by increased cytoplasmic Nrf2 levels and decreased nuclear Nrf2 levels.More importantly, Nrf2 was reactivated and tended to normalize after CA treatment (Fig 3A -3C).Then we investigated whether CA-induced Nrf2 activation further facilitated HO-1 expression, an Nrf2 target gene.Fig 3D -3F showed that DOX treatment suppressed HO-1 expression at the mRNA and protein levels in H9c2 cells, whereas the effect was blocked by CA in a time-dependent manner.The regulatory roles of CA in Nrf2/HO-1 signaling were further validated in animal models.Although CA did not restore the total Nrf2 levels in DOX-treated rats (Fig 4A and  4B), CA accelerated the nuclear translocation of Nrf2 (Fig 4C and 4D).Moreover, HO-1 expression was restored by CA in DOX-treated rats (Fig 4A and 4B).

CA increased cardiomyocyte viability by repressing ferroptosis
Nrf2 signaling is closely correlated with ferroptosis [40], which exerts an important role in myocardial oxidative injury [41,42].To assess the role of DOX and CA in ferroptosis, H9c2 cells were treated with DOX and CA, and then ferroptosis markers (iron concentration, ROS, MDA, and GSH levels) were examined.

Discussion
Despite its efficacy against many malignancies, DOX exhibits severe cardiotoxicity, affecting approximately 30% of patients within five years after treatment [43].Given that heart failure is a dominant reason for non-cancerous death after DOX treatment [43], relieving or eliminating DOX-triggered myocardial injury is essential to increasing its clinical application.Herein, we revealed the effect of CA on relieving DOX-induced cardiotoxicity.The main findings of this study were that: i) CA attenuated DOX-induced myocardial injury; ii) CA attenuated DOXinduced myocardial oxidative stress; iii) CA re-activated Nrf2/HO-1 signaling; and iv) CA enhanced cardiomyocyte viability by repressing cardiomyocyte ferroptosis.
Hydroxyl radicals, produced in physiological and pathological situations, are detoxified by GSH, which is a necessary antioxidant to maintain redox homeostasis through recycling enzymatic (GPXs, SOD, GST) and non-enzymatic (vitamin E) antioxidants [44].DOX frequently results in GSH depletion through dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) [44].NADPH is applied as the substrate of NADPH oxidases (NOXs) to generate ROS following DOX treatment [45].Given that DOX-induced ROS overgeneration is a vital pathogenic event in cardiomyocyte injury [46], the roles of CA in alleviating DOXinduced oxidative stress and subsequent ROS-triggered cardiomyocyte ferroptosis were investigated.In the study, we demonstrated that DOX treatment increased ROS levels in H9c2 cells, whereas CA repressed DOX-induced ROS.Furthermore, DOX decreased SOD, GSH, and GSH-Px levels in the heart tissues of DOX-treated rats and increased MDA levels, whereas the effect was blocked by CA, indicating that CA effectively represses DOX-triggered myocardial oxidative stress in vitro and in vivo.CA further mitigated DOX-triggered cardiomyocyte ferroptosis.These results suggest that CA is a promising agent for decreasing the side effects of DOX by inhibiting ferroptosis.
Mounting studies have revealed the biological role of plant-derived natural compounds in regulating ROS production.Emodin, an anthraquinone extracted from rhubarb, exhibits a protective role against myocardial infarction through suppressing ROS generation [47].Allicin sensitizes hepatocellular carcinoma (HCC) cells to 5-FU by further increasing ROS levels in cancer cells [48].As an antioxidant, allicin relieves trastuzumab-induced cardiotoxicity by decreasing ROS-mediated myocardial cell apoptosis [49].Astragaloside IV, an active component in Astragalus membranaceus, attenuates adriamycin-induced myocardial fibrosis through repressing cardiac ferroptosis and ROS levels [50].
The above results suggest that plant-derived natural ingredients possess the potential to relieve DOX-triggered myocardial injury by inhibiting DOX-induced oxidative stress.Given the biological role of CA in increasing Nrf2 nuclear translocation [27,28] and repressing oxidative stress [28], we explored whether CA can alleviate DOX-induced cardiotoxicity through decreasing myocardial oxidative stress.The current results demonstrated that CA relieved DOX-induced myocardial oxidative stress by assessing ROS, MDA, SOD, and GSH levels.Mechanistically, CA accelerated the nuclear translocation of Nrf2 and increased HO-1 expression in cardiomyocytes.Furthermore, we found that CA repressed DOX-induced cardiomyocyte ferroptosis, while Erastin treatment destroyed the effect of CA on increasing cardiomyocyte viability.The major limitations of this study were that, i) besides ferroptosis, other ROS-triggered cell death forms (apoptosis, pyroptosis, and necrocytosis) were regulated by CA, and ii) it is essential to further investigate how Nrf2 signaling was activated by CA.

Conclusions
These results suggest that CA alleviates DOX-induced cardiotoxicity, providing a promising opportunity to increase the clinical application of DOX.

Fig 1 .
Fig 1. CA alleviated DOX-induced myocardial injury in vitro and in vivo.H9c2 cells were treated with different doses of DOX (A) or CA (B), and then cell viability was measured using CCK8 after 24 h (n = 3).(C) H9c2 cells were treated with 4 μM of DOX and 100 μM of CA for 24 h, and cell viability was measured using CCK8 after 24 h (n = 3).(D and E) H9c2 cells were treated with 4 μM of DOX and 100 μM of CA for 24 h, and cell death was measured using TUNEL (n = 3).Rats were treated with 15 mg/kg of DOX with or without CA (50 mg/kg, 6 weeks), and serum CK (F) and LDH (G) levels were assessed using commercial kits (n = 5).(H) HE staining was used to assess myocardial injury in rats treated with DOX and CA (n = 5).(I) Masson staining was used to assess myocardial fibrosis in rats treated with DOX and CA (n = 5).qRT-PCR was performed to assess Col1α1 and α-SMA (J) mRNA levels in rats treated with DOX and CA (n = 5).*p<0.05,**p<0.01.https://doi.org/10.1371/journal.pone.0292124.g001 Fig 2A and 2B revealed that DOX obviously increased ROS levels in H9c2 cells, whereas CA repressed DOX-induced ROS.In a rat model of DOX-induced myocardial injury, DOX treatment suppressed SOD, GSH, and GSH-Px levels and increased MDA levels in cardiac tissues, whereas the effect was blocked by CA (Fig 2C-2F).These data suggest that CA effectively represses DOX-triggered myocardial oxidative stress in vitro and in vivo.

Fig 4 .
Fig 4. CA activated Nrf2/HO-1 signaling in vivo.(A and B) The total protein levels of Nrf2 and HO-1 in different groups of cardiac tissues (n = 5) were assessed using western blot assay.(C and D) The nuclear protein levels of Nrf2 in different groups of cardiac tissues (n = 5) were assessed using western blot assay.https://doi.org/10.1371/journal.pone.0292124.g004